This application is the national phase of international application PCT/BR97/00086 filed Dec. 30, 1997 which designated the U.S.
The present invention refers to the general field of the technology of DNA recombinant proteins, for the production of the recombinant growth factor (BEGF) of the virus BEAN 58058, to be used in human or veterinarian clinical practices or in research.
Growth is a primary, characteristic and fundamental process to any live organism. Although it is usually associated to the initial stages of life, growth is present as a normal property through adult life. This characteristic is shown mainly in the regenerative activities and in the process of programmed recycling of certain cellular types (James, R. and Bradshaw, R. A., Ann. Rev. Biochem. 53: 259-292,1984).
Several factors contribute to stimulating and controlling growth. Among these factors are hormones, neural growth factor, contact to proximal homologous or heterologous cells, interaction with extracellular structures (extracellular matrix), and others. Among the several agents involved in the process of growth regulation a group of proteins became particularly prominent in the last years, being recognized initially as hormones. However, the more careful analysis of these factors demonstrated that they possessed their own characteristics and they came to be studied in a group that was called polypeptide growth factors or simply PGFs. Several characteristics differentiated these proteins from the hormones. Among them was the fact that they are eminently protein molecules. PGFs are transported from the cell through tissue diffusion, following an autocrinous and paracrinous secretion model. To the contrary, hormones present an endocrine secretion model, where the molecules are stored in vesicles and released in the blood stream (James, R. and Bradshaw, R. A., Ann. Rev. Biochem. 53: 259-292, 1984).
The first isolated PGF was the nervous growth factor (NGF) that acts by stimulating the division and differentiation of sympathetic nerves during the development of vertebrates. Another important member of the family of PGFs was discovered, soon after, in consequence of the study of the activity of NGF in mice. This new element was denominated epidermal growth factor (EGF) and it was detected by stimulating the growth of epidermal and epithelial cells. Several other growth factors were described, among them were the growth factors similar to insulin (IGF I and II ), interleukin II (or growth factor of T cells), growth factor derived from platelets (PDGF), transforming growth factor (TGF) erythropoeitin, and others (James, R. and Bradshaw, R. A., Ann. Rev. Biochem. 53: 259-292, 1984; Carpenter, G., Ann. Rev. Biochem. 56: 881-914, 1987; Hillman, R. S. 1990. Hematopoietic agents: Growth factors, minerals and vitamins. in: Gilman, A. G. ;Rall, T. W. ;Niens, A. S. and Taylor, P. (ed.) The pharmacological basis of therapy. 8th edition. Louis Sanford, N.Y., USES., cap. 14, p. 277-1310, 1990).
This superfamily of proteins includes elements structurally similar but with different physiologic functions. The main characteristic is that all these factors possess mitogenic and intrinsic regulatory activities, coordinating the cellular growth. In fact, each PGF possesses specific receptors, located in the plasma membrane of sensitive cells (James, R. and Bradshaw, R. A., Ann. Rev. Biochem. 53: 259-292, 1984). However these concepts are being modified, since today it is known that the receptors of EGF, for example, are present in a wide variety of cellular types, in different tissues. Besides, it is known that the receptors of EGF are responsible for the biological activity of at least two other growth factors similar to EGF: the transforming growth factor (TGF - xcex1) and the growth factor of the vaccinia virus (VGF). The last is a molecule encoded in the genome of the vaccinia virus. Its function is probably associated with the mitotic proliferation of infected and non-infected cells. VGF is responsible for the proliferation activity of the cells through its binding to the receptors in these cells. This phenomenon, well known as molecular mimicry, would create favorable conditions for viral multiplication (Buller, R. M. and Palumbo, G. J., Microbiol. Rev., 55:80-122, 1991; Alamo, A. and Smith, G L, Immune. Today, 16: 474-478, 1995).
The precursor polypeptides of EGF and TGF - xcex1 present peptide signal and transmembrane amino acid sequences, suggesting that these factors are located at the cytoplasmic membrane before the cleavage that originates mature molecules. In the same way, the precursor of the VGF protein carries a hydrophobic amino acid in the amino and carboxy terminal that would work as peptide signal and transmembrane sequences respectively (Stroobant,P. et al., Cell, .42: 383-393, 1985). VGF, EGF and TGF - xcex1 present groups of residues, mainly cysteines, highly conserved in the mature proteins. These residues, located in homologous sites in these growth factors, are responsible for the formation of three disulfide bridges, which are responsible for the tertiary structure of the molecules and, consequently, for its similar biological actions (Twardzik, D. R. et al., Proc. Natl. Acad. Sci. It USES, 82: 5300-5304, 1985).
The growth factor of the virus BeAn 58058 (BEGF) presents amino acids sequences very similar to the sequence of VGF. There are modifications in the amino acid 17 and 78 in relation to the sequence of VGF. These modifications are changes of a leucine to a serine and an asparagine to lysine, respectively, in the polypeptide sequence of BEGF.
During the 1980""s, the physiologic role of PGFs became questioned, once many of these factors became related with processes of cellular transformation and tumor development. PGFs that were initially related to the cellular transformation are EGF, PDGF and mainly the group of TGFs.
Nevertheless. later authors began to evaluate the hypothesis of the therapeutic use of PGFs essentially based on their mitotic properties. The possibility of the therapeutic use of these factors was tested firstly and used in the treatment of hematological diseases. That is the case of the erythropoeitin and of GM-CSF. Such factors have also been used as adjuvant in the treatment of serious infections and in patients submitted to chemotherapy with immune-suppressor drugs. The erythropoeitin is related to the erythropoiesis and, in its absence, the patient has serious anemia. The clinical use of GM-CSF has been tested with success in patients with neutropenia after neoplasia, congenital neutropenia, aplastic anemia, myelodysplastic syndromes and AIDS. GM-CSF acts to increase the granulocyte and macrophages proliferation (Hillman, R. S. 1990. Hematopoietic agents: Growth factors, minerals and vitamins. in: Gilman, A. G. ;Rall, T. W. ;Niens, A. S. and Taylor, P. (ed.) The pharmacological basis of therapy. 8th edition. Louis Sanford, NY, USES., cap. 14, p. 277-1310, 1990).
The therapeutic use of PGFs is still very restricted and their use is still limited to the experimental field. However, the study of growth factors such as EGF, PDGF, IGFs and TGFs open perspectives for the treatment of several illnesses, whose cure depends on tissue regeneration. In fact, Matsuda, N et al. (J. Periodontol. 63:515-525, 1992), tested with efficiency the action of several PGFs (EGF, PDGF, IGF-I and TGF -xcex1) in the proliferation of mouse periodontal fibroblasts. Additionally, an important increase in the synthesis of collagen of these cells was observed. These data evidence the possibility of the use of such factors in the treatment of periodontal diseases.
The topical administration of EGF in small wounds caused by burns or incisions accelerates the epidermal regeneration through the stimulation of the keratinocytes, phenomenon necessary for the healing process. In the same way, the topical administration of TGF-xcex1 and VGF accelerated the regeneration of wounds caused by burns of second degree. Additionally, the treatment with TGF-xcex1 and VGF was shown to be more efficient than the treatment with EGF, mainly when comparing the amounts used (Schultz et al Science, 235: 350-352, 1987.)
These results illustrate the clinical potential of the several well-known growth factors and they reflect the optimistic perspectives of the use of PGFs in other pathological processes that need inductors of cellular proliferation and regulatory elements of growth.
It is an object of the present invention to describe the recombinant BeAn 58058 growth factor (BEGF) protein isolated from virus BeAn58058, their corresponding encoding recombinant DNA molecule and the process of production of the recombinant BEGF protein produced through techniques of genetic engineering, to be used in human or veterinarian clinical practices or in research.